Tissue culture is a method of biological research where plant tissues are grown under sterile conditions. There are several methods of plant tissue culture including seed culture, embryo culture, callus culture, cell culture, bud culture, meristem culture, and protoplast culture. Each method involves explants from various plant tissues being placed on nutrient media to induce cell growth and differentiation. Tissue culture has many applications including rapid clonal propagation, inducing genetic variation, producing transgenic plants, and conserving plant genetic resources.

1. Tissue culture
•“… A method of biological research in which fragments
of tissues from an animal or plant are grown in vitro in
artificial medium under aseptic conditions and continue
to and function.”
•“… the aseptic culture of plant protoplasts, cells, tissues
or organs under aseptic conditions which lead to cell
multiplication or regeneration of organs or whole
plants.”

2. Methods of plant tissue culture
Methods of plant tissue culture
culture

3. Embryo
culture
Cell
culture
Callus
culture
Seed
culture
Bud
culture
Meristem
culture
Protoplast
culture
Organ
culture
Methods of
plant tissue
culture

4. Seed culture
• Seed culture is an important technique when explants are taken from in vitro-
derived plants and in propagation of orchids.
• Sterilising procedures are needed for plant materials that are to be used directly,
as explant source can cause damage to tissues and affect regeneration.
• In that case, culture of seeds to raise sterile seedling is the best method. Orchid
cloning in vivo is a very slow process.

5. Embryo Culture
• This involves the in-vitro development of an embryo. For this, an embryo is isolated
from a living organism. Both, a mature or an immature embryo can be used in the
process.
• Mature embryos can be obtained from ripe seeds. The immature embryos are obtained
from the seeds that failed to germinate. The ovule, seed or fruit is already sterilized,
therefore, it does not need to be sterilized again.
i. Mature embryo culture:
• It is the culture of mature embryos derived from ripe seeds. This type of culture is done
when embryos do not survive in vivo or become dormant for long periods of time or is
done to eliminate the inhibition of seed germination.
ii. Immature embryo culture/embryo rescue:
• It is the culture of immature embryos to rescue the embryos of wide crosses. This is
mainly used to avoid embryo abortion with the purpose of producing a viable plant.
The underlying principle of embryo rescue technique is the aseptic isolation of embryo
and its transfer to a suitable medium for development under optimum culture
conditions.

7. Callus Culture
• Callus is basically a more or less non-organised tumor tissue which usually arises
n wounds of differentiated tissues and organs.
• Thus, in principle, it is a non-organised and little differentiated tissue. The cells in
callus are of a parenchymatous nature.
• When critically examined, callus culture is not homogeneous mass of cells,
because it is usually made up of two types of tissue: differentiated and non-
differentiated.
• Callus formation takes place under the influence of exogenously supplied growth
regulators present in the nutrient medium.
• The type of growth regulator requirement and its concentration in the medium
depends strongly on the genotype and endogenous hormone content of an
explant.

9. Organ Culture:
• It is an isolated organ grown in vitro. It can be given different names depends upon the
organ used as an explant. For example, meristem or shoot tip culture, root culture,
nucellus culture, endosperm culture, ovule culture, a culture for production of
androgenic haploids while ovule and ovary culture vitro production of gynogenic
haploids. The culture of plant results in three types of in vitro culture.
Organised:
• The culture of whole plants (embryos, seeds) and organ has been termed as organised
culture. In this, characteristic organised structure of a plants individual organ is
maintained.
Non-organised:
• If cells and/or tissues are isolated from an organised part of a plant, dedifferentiate and
are then cultured, a non-organised growth in the form callus tissue results.
Non-organised/organised:
• This type of culture is intermediate between the above two types. Cells in an isolated
organ or tissue first dedifferentiate and then form tissues which then re-differentiate to
form organs (roots or shoots) or embryos.

11. PROTOPLAST CULTURE
• Protoplast is defined as naked plant cells or plant cells without a
cell wall. It consists of plasmalemma containing all the other
cellular content or components in it.
• In tissue culture labs it’s used to regenerate a whole plant
providing suitable artificial medium and environmental
conditions. This process is known as protoplast culture.
• Cooking for the first time isolated protoplasts of plant tissue by using
cell wall degrading enzymes viz. cellulase, hemicellulase, pectinase,
and protease extracted from a saprophytic fungus Trichoderma viride.
Now the protoplasts are cultured in vitro. Sterilisation of the leaf
samples with sodium hypochlorite solution.

13. Anther Culture:
• Anther culture is the process of using anthers to culture haploid plantlets.
• The technique was discovered in 1964 by Guha and Maheshwari.
• This technique can be used in over 200 species, including tomato, rice, tobacco,
barley, and geranium.
• Some of the advantages which make this a valuable method for obtaining haploid
plants are: the technique is fairly simple it is easy to induce cell division in the
immature pollen cells in some species a large proportion of the anthers used in
culture respond (induction frequency is high) haploids can be produced in large
numbers very quickly.

15. Cell culture
• Cell culture refers to the removal of cells from an animal or plant and their
subsequent growth in a favorable artificial environment.
• The cells may be removed from the tissue directly and disaggregated by
enzymatic or mechanical means before cultivation, or they may be derived from a
cell line or cell strain that has already been already established.
Primary Culture
• Primary culture refers to the stage of the culture after the cells are isolated from
the tissue and proliferated under the appropriate conditions until they occupy all
of the available substrate (i.e., reach confluence).
• At this stage, the cells have to be subcultured (i.e., passaged) by transferring
them to a new vessel with fresh growth medium to provide more room for
continued growth.

17. Bud Cultures
• The plant buds possess quiescent or active meristems depending on the physiological
state of the plant.
• Two types of bud cultures are used— single node culture and axillary bud culture.
Single node culture:
• This is a natural method for vegetative propagation of plants both in vivo and in vitro
conditions.
• A bud along with a piece of stem is isolated and cultured to develop into a plantlet.
• Closed buds are used to reduce the chances of infections.
• In single node culture, no cytokinin is added.
Axillary bud culture:
• In this method, a shoot tip along with axillary bud is isolated.
• The cultures are carried out with high cytokinin concentration.
• As a result of this, apical dominance stops and axillary buds develop.
• Good axillary bud culture – cytokinin/ auxin ratio is 10:1.

19. Meristem Culture
What is Meristem?
• Meristem is a part of the plant, which plays a key function to increase the plant
length.
• It possesses meristematic cells that are continuous, oval, polygonal and rectangle
in shape. There is no intercellular space between the meristematic cells.
• Meristem culture is defined as the tissue culture technique, which uses apical
meristem with 1-3 leaf primordia to prepare clones of a plant by the vegetative
propagation.
• This technique primarily involves the isolation of meristem by applying a V-Shape
cut in the stem.
• By the culturing of shoot meristem, adventitious roots can be regenerated in this
method.
• The shoot tip’s preferable size is 10 mm for the generation of the virus-free plant,
but the shoot tip’s size does not matter in vegetative propagation.

21. Applications of Tissue Culture
1. Rapid Clonal Propagation
2. Soma-clonal Variation
3. Transgenic Plants
4. Induction and Selection of Mutations
5. Resistance to Weedicides.
6. Genetic transformation
7. Genetic resource conservation
8. Pathogen Eradication
9. Somatic hybridization
10.Haploid and Doubled haploids Production
11. Agriculture
12. Germplasm conservation
13. Horticulture and Forestry
14. Industries

22. 1. Rapid Clonal Propagation
• A clone is a group of individuals or cells derived from a single parent
individual or cell through asexual reproduction.
• All the cells in callus or suspension culture are derived from a single
explants by mitotic division.
• Therefore, all plantlets regenerated from a callus/suspension culture
generally have the same genotype and constitute a clone.
• These plantlets are used for rapid clonal propagation.

24. 2. Soma-clonal Variation:
• Genetic variation present among plant cells of a culture is called
soma-clonal variation.
• The term soma-clonal variation is also used for the genetic variation
present in plants regenerated from a single culture.
• This variation has been used to develop several useful varieties.

26. 3. Transgenic Plants:
• A gene that is transferred into an organism by genetic engineering is
known as transgene.
• An organism that contains and expresses a transgene is called
transgenic organism.
• The transgenes can be introduced into individual plant cells.
• The plantlets can be regenerated from these cells.
• These plantlets give rise to the highly valuable transgenic plants.

28. 4. Induction and Selection of Mutations:
• Mutagens are added to single cell liquid cultures for induction of
mutations.
• The cells are washed and transferred to solid culture for raising mu
ant plants.
• Useful mutants are selected for further breeding.
• Tolerance to stress like pollutants, toxins, salts, drought, flooding etc.
can also be obtained by providing them in culture medium in
increasing dosage.
• The surviving healthy cells are taken to solid medium for raising
resistant plants.

30. 5. Resistance to Weedicides:
• It is similar to induction of mutations.
• Weedicides are added to culture initially in very small concentrations.
• Dosage is increased in subsequent cultures till die desired level of
resistance is obtained.
• The resistant cells are then regenerated to form plantlets and plants.

32. 6. Genetic transformation
• Genetic transformation is the most recent aspect of plant cell and tissue
culture that provides the means of transfer of genes with desirable trait
into host plants and recovery of transgenic plants.
• The technique has a great potential of genetic improvement of various crop
plants by integrating in plant biotechnology and breeding programmes.
• It has a promising role for the introduction of agronomic important traits
such as increased yield, better quality and enhanced resistance to pests
and diseases (Sinclair et al., 2004).
• Genetic transformation in plants can be achieved by either vector-
mediated (indirect gene transfer) or vector less (direct gene transfer)
method

34. 7.Genetic resource conservation
• Conservation of plant genetic resources is necessary for food security and
agro-biodiversity which need better use of a broader range of genetic
diversity across the globe.
• Genetic diversity provides options to develop through selection and
breeding of new and more productive crops, resistant to biological and
environmental stresses.

35. 8.Pathogen Eradication
• Crop plants, especially vegetatively propagated varieties are generally infected
with pathogens.
• The most significant advantages offered by micro propagation are large numbers
of disease free propagules can be obtained from a single plant in a short period,
propagation can be carried out throughout the year and the propagating
material can be accommodated in a small space, reduction of labor costs for
germplasm maintenance, avoidance of field inspections and environmental
hazards, easy availability of material for micro propagation and rapid
multiplication.

37. 9. Somatic hybridization
• Somatic hybridization (SH) via protoplast fusion is an important tool for the
production of interspecific and intergeneric hybrids and involves the fusing
protoplasts of two different genomes followed by the selection of desired
somatic hybrid cells and subsequent regeneration of hybrid plant.
• It is efficient mean of gene transfer from one species to another so as to break
the crossing barriers and integration of parental nuclear and cytoplasmic
genomes.
• SH has been widely exploited in different horticultural crops to create novel
hybrids with increased yield and resistance to diseases.
• In addition, it has also been used for salt tolerance, quality improvement,
transfer of cytoplasmic male sterility (CMS), seedless triploids and rootstock
improvement

39. 10.Haploid and Doubled haploids Production
• Breeders have used different methods to fix and develop homozygous
genotypes through isolation of homozygous and homogeneous
genotypes through conventional inbreeding methods which take
several cycle inbreeding and selection and this also may not produce
true homozygous line.
• However, now a day, plant tissue culture advanced and haploid and
double haploid production through anther and ovule culture have
been practicing.

41. 11. Agriculture
• Anther & pollen culture-Production of haploid plants.
• Production of virus-free plants for safe germplasm transfer.
• Screening of in vitro lines for stress and disease resistance.

42. 12. Germplasm conservation
• An extension of micropropagation techniques through two methods:
• Slow growth techniques e.g.: ↓ Temp., ↓ Light, media supplements ( growth
retardants).
• Medium-term storage (1 to 4 years)
Cryopreservation.
• Ultra low temperatures in liquid nitrogen at -196 0C.
• Stops cell division & metabolic processes
• Very long-term (indefinite?)

43. 13. Horticulture and Forestry
• Micropropagation method is used for rapid multiplication of ornamental plants as
well as important trees yielding high fuel, pulp, fruits or oil at a large scale.
• Today's high yielding oil palms on plantations in Malaysia were generated in the
1960s by tissue culture methods.
• These palms produce 30% more oil than normally cultured palms. Improvement
of economically important forest trees is being done through genetic
transformation and rapid Micropropagation .
• e.g in vitro regeneration and genetic transformation of conifers.

44. 14. Industries
• Plant cell culture is used for biotransformation (modification of functional groups
of organic compounds by living cells).
• Food and agricultural biotechnologists are involved in using tools of molecular
biology to enhance the quality and quantity of foods and economic crops.
• For example, Golden Rice was genetically enhanced with added beta carotene,
which is a precursor to Vitamin A in the human body.
• Plant cells can be cultured in fermenters for the industrial production of
secondary Metabolites using cell culture.